Conceptus Development: Exosomes & Implantation

Fertilisation and Development of the Conceptus

The Role of Exosomes in Early Embryonic Development

• From zygote to blastocyst development stages include 2-cell, 4-cell, 8-cell, morula, and blastocyst
• Exosomes play a role, Oc-Exo/miRNAs, in altering transcription, increasing birth rates, decreasing apoptosis, and improving differentiation.
• From the uterus to the blastula, exosomes promote embryo implantation by targeting miR-30d.
• Let-7 targeting C-MYC/mTORC1 and mTORC2 in exosomes induce embryonic diapause.
• Exosomes from blastula to the uterus inhibit embryo adhesion to guarantee dissociation via miR-661
• LINC00478 and ZNF81 are targeted to reduce transcription levels
• Exosomes from the inner cell mass (ICM) to the trophectoderm (Tc) in the blastula promote blastocyst development through laminin and fibronectin.

Development of Conceptus in the Uterine Tube

• Sperm are sequestered in the isthmus
• Sperm that traverse the female genital tract are sequestered in the isthmus of the uterine tubes.
• Sperm are released and migrate to the ampullae of the tubes where fertilization occurs at the start of ovulation.
• Sperm ascent and oocyte descent to the ampullae are synchronized.
• Fertilization requires the fusion of nuclear material from one spermatozoon and one oocyte
• The oocyte has mechanisms to prevent fertilization by more than one spermatozoon (polyspermy), as it results in abnormal embryo development; this block mechanism is called "polyspermic."
• After fertilization, cleavage of the zygote begins.
• Peristaltic contractions and ciliary currents propel the zygote towards the uterus
• When it reaches the uterus, the zygote consists of 16 to 32 cells in the form of the morula.
• Travel time in cattle averages 1.3 to 4 days; in dogs and cats, it's 2.5 to 8 days.
• After morula, the conceptus becomes a blastocyst
• Morula becomes hollowed out to form a blastocyst, its wall becomes two layered.
• Inner layer becomes endoderm and outer is the ectoderm.
• Cavity is known as blastocoele.
• Embryo forms from a thicker portion of the ectoderm referred to as the embryonic knot or disc.
• Remaining portion of the ectoderm is referred to as the trophoblast, which has an important role in placenta formation.
• Fertilized egg of the ewe enters the uterus at the eight-cell stage on day 3
• In sows, the conceptus progresses down the uterine tube within 2 days of ovulation, arriving in the uterus as a four-to-eight-cell stage.
• Tubal transport of the egg in mares takes 5 to 6 days, reaching the blastocyst stage.
• Unfertilized equine eggs remain in the tubes for months and slowly degenerate.
• The duration of travel by the fertilized egg in the uterine tube is determined positively by the degrees of activity of the tubal muscle and cilia.
• Duration of travel can be negatively impacted by the muscular constriction at the tubal isthmus or at the uterotubal junction.
• Both positive and negative factors are influenced by variable concentrations of ovarian steroid hormones and by locally produced prostaglandins.

Development of Conceptus in the Uterus

• From the arrival time in the uterus until attachment, the zygote is propelled or aspirated into the uterine lumen, where it is nourished by the exocrine secretions of the uterine glands known as "uterine milk."
• In polytocous species, blastocysts are distributed throughout the uterus to utilize the space effectively, thus, free migration of embryos occurs between the cornua, regardless of the side of ovulation.
• Monotocous cows rarely experience migration
• Migration is not uncommon in ewes
• In the mare, extensive migration always occurs.
• A characteristic feature of the elongated porcine blastocyst is that it aligns with the uterine attachment of the broad ligament
• Embryonic attachment timeframe to the uterus for cows is 12 days, sows 18 days, ewes 15 days, dogs and cats 13 to 17 days, and mares 35 to 40 days.

Fetal Membranes and Placenta Formation

• Knowledge of the formation and structure of the fetal membranes is crucial for correct interpretation by manual palpation.
• It is also crucial for visual interpretation of ultrasound images of early pregnancy
• Early embryonic structures are involved in the dialogue between the embryo and the uterus, enabling maternal recognition of pregnancy, implantation, placentation, and growth of the conceptus.
• Around the time of the blastocyst hatching, embryonic (subsequently fetal) membranes develop, Some have a major role in the formation of the placenta and include: the yolk sac, amnion, chorion, and allantois

Yolk Sac

• After hatching, the inner (endodermal) layer of cells of the bilateral blastocyst migrate around the inner surface of the ectoderm and blastocoele to form it .
• In mares, the yolk sac completely forms by day 11
• Yolk sac fluid of the equine embryo is the first easily identifiable structure during ultrasonographic pregnancy diagnosis
• Equine yolk sac fluid is hypertonic and contains high levels of fructose, proteins, and prostaglandins
• The wall of the yolk sac produces IGF-1 and synthesizes and metabolizes steroids and prostaglandins.
• In mares, dogs, and cats, the yolk sac persists longer and can be easily recognized during ultrasonic scanning

Amnion

• The amnion develops as an enveloping fold of ectoderm and mesoderm that ultimately fuses and completely surrounds the embryonic disc, embryo and fetus
• The main role of the amnion is protective, containing the amniotic fluid in which the embryo "floats" and moves.

Chorion

• The chorion is formed by the outer layer of mesoderm and trophoblast

Allantois

• The allantois is the last of the embryonic/fetal membranes to form, developing as a thin-walled sac-like outgrowth of endoderm surrounded by mesoderm from the embryonic gut, close to the opening connecting the gut with the yolk sac.
• Mesoderm covering the allantois fuses with the mesoderm lining the chorion to form the allantochorion (chorioallantois), a structure involved in placenta formation.
• The allantois fuses with the amnion to form the allantoamnion
• early development of the ruminant embryo causes extensive fusion between the allanto-amnion and allantochorion, or "amnio-chorion."
• Recognition of allantoic fluid enables early ultrasonographic pregnancy diagnosis between days 20 and 25.
• By day 35 on wards, the allantoic sac begins to distend the gravid uterine horn, and at this stage, it can be palpated manually together with the amniotic vesicle

Equine Placentation

• Equine conceptus does not show initial rapid elongation of the blastocyst – chorion as in ruminants and pigs
• At 35 days, the equine chorion is oval and distended by the more allantoic fluid; it causes more discrete uterine enlargement in the cow
• In mares, unlike cows, the amniotic and allantoic sacs do not fuse as in ruminants.
• The amnion floats freely and is contained within the allantoic sac, except at the umbilicus attachment throughout gestation
• Starting around day 40, there develops and increases the complexity over the next 100 days of multibranded interdigitations with the endometrium to form microcotyledons.
• Microcotyledons are the fundamental unit of the fetal-maternal interface in the equine placenta.
• The microcotyledons consist of a cluster of highly vascularized chorionic villi that extend into elaborate invagination of the endometrium
• Microcotyledons are distributed over the entire surface of the allantochorion and can be seen by the naked eye, giving allantochorion a stippled or velvety appearance
• The trophoblast of the conceptus separates into its invasive and non-invasive components by day 35
• Thickened annulleted chorionic girdle invade the endometrium to form the endometrial cups- a structure unique to equines.
• Endometrial cups form during pregnancy and are the source of equine chorionic gonadotropins (eCG).
• The placenta-associated structure is derived from fetus and aims to increase mare's immunological tolerance in order to shield the developing foal
• The endometrial cups are present from the 6th to the 20th week of gestation

Horse-specific structures

• eCG, equine chorionic gonadotropins, is produced, stimulating strong maternal, humoral, and cell-mediated immune responses, ultimately curtailing its lifespan.
• Hippomanes are attached to the allantochorion or float in the allantoic fluid; they're calculi of cellular and inorganic debris.
• Hippomanes is a palm-sized or larger, olive to light tan, rubbery consistency slab of tissue typically found with the placenta at foaling.
• This feature is composed of various fetal waste products accumulating over the pregnancy.

Pig Placentation

• After the blastocyst hatches at day 7, they enlarge to be 8 to 10mm, flaccid spheres
• The conceptus is distributed and spaced along both horns before adherening on day 12 to the endometrium near the broad ligament attachment

Dog and Cat Placentation

• Fixation and implantation begin by days 17 to 19 in dogs
• Until 20 days: there is a choriovitelline placenta in which vascular yolk is involved.
• At 24 days, the yolk sac is three times the length of the embryo and can be recognized during ultrasonic scanning.
• Around 20 days: develops allantois.

Types of Placentation

• Placentation is classified by villi arrangement and how maternal tissue comes into contact with the chorion.
• Diffuse placentation -villi dispersed uniformly in the mare and sow.
• Cotyledonary placentation -villi grouped in multiple circumscribed areas like in the ruminant.
• Zonary placentation - villi arranged a broad encircling belt
• Classified according to maternal vs fetal tissue presence after birth, resulting in either deciduae transfer(bitch and cat) or non-deciduae (no transfer)
• According to the degree of proximity of the maternal and fetal blood circulation
• Recognizes the phagocytic property of the trophoblast or chorionic epithelium exerted on contact tissues
• In epitheliochorial placentation, such as in horses and pigs, the chorion comes into contact with the endometrium, and no maternal tissue is lost during placenta formation. The ruminant is synepitheliochorial, featuring a syncytium on the maternal side due to fusion derived from the trophectoderm with uterine epithelial cells.

Other Classifications

• Syncytium-only temporarily forms in bovine placentomes; it is not temporary in sheep and goats
• Endotheliochorial placentation happens through further invasion into the endometrium. This is typical of carnivores.
• Haemochorial placentation, the tissues separate the fetal and maternal blood and are typical of the carnivora
• Blood pigment deposits (haematomata) appear at margin, transfers iron to fetus.

Other Factors

• Degree of intimacy of the maternal and fetal placental blood vessels is the basis for the “placental barrier" variable function across species
• Placenta, the dynamic organ, is responsive to the nutritional needs of the fetus for growth and development during intrauterine development

Fetal Fluids

• Types of foetal fluid include amniotic and allantoic fluids
• During the first third of pregnancy, allanoic fluid predominates. Amniotic fluid predominates during the second trimester. Finally, during the later part of pregnancy more allanoic fluid is present
• Throughout gestation- allantoic is watery/urine-like First two thirds = amniotic fluid is similar to that
• Remainder of the period- mucoid in fluid
• Allantoic sac forms 1st water bag. and Amnion forms the 2nd water bag during the first third.

Relationships Between Fetal Membranes of Twins and Multiple Fetuses

• Distal pole of one allantochorion invaginates the proximal extremity of the other, as with mares pregnant with twins and a lack of uterine strength.
• This leads to the unequal sharing, thus the competition for the reduced endometrium to which the trophoblast attaches to form the placenta
• Usually one fetus suffers severe growth retardation, leading to death, failure of pregnancy, or birth live.
• Intervening walls can cause, forrowing uterine contractions and/or a continuous allantochorionic tube through which the fetuses pass during parturition (or synchorial tubes)

Fetal movements during pregnancy

• Fetal position, presentation is impacted by their movements that can be observed by domestic animals as early as day 40 via ultrasonography Active fetal movements change intrauterine presentation, position, and posture during birth.

Explore the conceptus development from zygote to blastocyst, focusing on the role of exosomes. Learn how exosomes influence transcription, birth rates, apoptosis, and differentiation as the conceptus moves from the uterus to the blastula. Discover the importance of miRNA-30d and miR-661 in embryo implantation and dissociation.